Defining the gene expression cascade underlying cell fate determination is a key element in understanding development. The availability of whole genome microarrays, coupled with the isolation of nearly pure cell type populations, allows one to begin to define the transcriptome associated with specific cell fates. Muscle cells have been attractive targets for such studies in mature animals due to their ease of isolation andor culture and their importance in human pathologies. It has been more difficult, however, to determine early in vivo embryonic myogenic gene expression patterns that would give insights into the regulation of muscle development. To this end, we have done a developmental profile of gene expression and identified key transcriptional regulators. Currently we are trying to understand the molecular mechanisms by which they regulate myogenesis. We previously showed that the factor PAL-1 is genetically upstream of two transcription factors needed for myogenesis (HLH-1 and UNC-120). In the current reporting period we demonstrated direct binding of PAL-1 to the promoter of the hlh-1 gene, identified important cis-acting regulatory sequences, and validated the function of these elements using wild type and mutant backgrounds. This provided the first molecular evidence for the transcriptional cascade initiating muscle development in the C. elegans embryo. We have begun to extend these studies to other muscle lineages in the embryo that are not dependent on PAL-1 with the goal of defining novel myogenic factors and understanding their deployment during early development. We also continue to study the in vivo target genes activated by the myogenic regulator, HLH-1,a homolog of the vertebrate MyoD family. We have previously shown that ectopic expression of hlh-1 in early C. elegans embryos is sufficient to convert most blastomeres to a body wall muscle like fate. To define the transcriptional targets of HLH-1 that underlie muscle cell fate specification and differentiation, we have use chromatin immunoprecipitation (ChIP) followed by probing whole genome tilling microarrays (Chip). The results are beginning to reveal the in vivo binding sites for this transcription factor, allowing us to correlate DNA binding with gene expression in the early embryo. Our hope is that this approach will allow a more exhaustive description of the transcriptional cascade that plays out during muscle cell development.